This study evaluated the effect of three different silanes and post-silanization treatments on the retentive strength of fiber posts luted with an etch-and-rinse resin cement.One hundred intact maxillary central incisors were randomly divided into 10 groups after endodontic treatment and post space preparation (n=10). The fiber posts were etched using 24% hydrogen peroxide. Posts of the control group did not receive silane. In nine experimental groups, each of the three silanes used, Scotchbond Universal adhesive, Bis-Silane and Porcelain Primer, was subjected to three treatments: air-drying at 25°C, warm air-drying and CO2 laser heat treatment. After cementation of the treated posts using One-Step Plus/Duo-Link cement, the specimens were stored for one weak and then subjected to pull-out bond strength (PBS) testing. The data in Newton (N) were analyzed using two-way ANOVA and Tukey tests (α=0.05).p<0.001). The interaction of the two factors was not statistically significant (p=0.15). The effect of Porcelain Primer on PBS was significantly higher than those of universal adhesive (p<0.001) and Bis-Silane (p=0.01), with similar results for the two latter. Warm air-drying and laser treatment significantly increased PBS (p<0.001). The lowest and highest PBS was obtained in the control (no silane) group (190.9±31) and laser-treated/ Porcelain Primer group (377.1±50), respectively.PBS was significantly affected by silane type and post-silanization treatment (Warm air-drying and CO2 laser heat treatment had a significantly beneficial effect on retentive strength of fiber posts. Porcelain Primer was significantly more effective than universal adhesive and Bis-Silane. Key words:Laser heat treatment, Pull-out bond strength, fiber post. Use of fiber-reinforced composite post (FP) has increased to provide retention for the final restoration in endodotically treated teeth . This isOrganosilanes, with an intrinsic dual reactivity, act as an adhesion promoter between the inorganic surface and polymeric resins through an organic functional group and three alkoxy groups ,5. FPs cet al.. This effect was attributed to surface warming induced by a focused CO2 laser beam at low energy setting (Carbon dioxide (CO2) laser is one of the most commonly used lasers in dental practice. The beneficial effect of CO2 laser beams on the performance of silane coupling agent and on improving bonding of resin to ceramic was reported by Chen  setting .Recently, a new category of adhesive systems was introduced as universal adhesives. In Scotchbond Universal, methacryloxydecyl phosphate monomer and silane coupling agent are combined, enabling bonding to different substrates such as silica-based ceramics . It mighTherefore, this study was designed to test the null hypothesis that CO2 laser treatment, compared to air- and warm air-drying and different silane types, has no effect on the retentive strength of FP cemented in the root canal.One hundred human maxillary central incisors with similar size and anatomic shape and straight roots without cracks, were selected and stored in 0.5% chloramines-T solution at 4°C until use. They were used following informed consent from patients and approval of the research protocol by the local Ethics Committee. The roots were sectioned to provide a uniform length of 12 mm, using a water-cooled diamond saw . They were embedded in epoxy resin blocks in the vertical axis and were endodontically treated.The specimens were stored in water for one week for the complete setting. Afterwards, a 5-mm-deep post space was prepared using a #2 drill from the respective post manufacturer by the same operator. Cleanliness of the root walls was confirmed by radiographs. Glass fiber posts  were tried in the post space for a passive fit in the prepared depth without a need for relining. Post surfaces were etched using 24% hydrogen peroxide for one minute, water-rinsed and air-dried.The FPs were divided into one control group and nine experimental groups (n=10), based on three silane types and three post-silanization treatments. No silane was applied in the control group. In groups 2-4, Scotchbond Universal adhesive (3M ESPE) was applied onto post surface for 20 sec and then air-thinned/dried for 10 sec at 25°C from a distance of 10 mm in the air-dried group. In the warm air-dried group, air-thinning/drying was performed at 50 ± 5°C using a blow dryer . In the laser-treated group, following air-thinning/drying, the post surface was subjected to the CO2 laser  heat treatment at 1 W in continuous mode from a distance of 30mm for 20 sec with a size tip diameter of 1 mm. In a preliminary study, the irradiation parameters used were tested to confirm the temperature produced (50 ± 5°C) on the post surface. In the three groups, the temperature used was checked by a laboratory thermometer  by placing the electrode on the post surface. Finally, the adhesive was cured for 10 sec with a light-curing unit  at 600 mW/cm2.In groups 5-7, two-bottle silane solution Bis-Silane (Bisco) was mixed and applied on the post surface according to manufacturer’s instructions.In groups 8-10, one-bottle silane Porcelain Primer (Bisco) was applied in two layers according to manufacturer’s instructions.Etch-and-rinse resin cement One-Step Plus/Duo-Link Universal (Bisco) was used to cement the treated posts. After acid etching and water rinsing, the root canals were blot-dried with paper points. Then One-Step Plus was applied according to manufacturer’s instructions. The mixed cement was applied into the post space and onto the post surface. The post was immediately seated with a slight vibratory motion and held under finger pressure for 10 sec. The excess cement was removed and light polymerization was carried out for 60 sec using VIP Junior light-curing unit at 600mW/cm2. The specimens were stored in distilled water at 37°C for one week.For pull-out testing, a tensile force was applied parallel to the long axis of both the post and root at a crosshead speed of 1mm/min in a universal testing machine  until the post was dislodged. At that moment, the maximum load was recorded in Newton (N). Data in all the groups were analyzed with one-way ANOVA and post-hoc Tukey tests. Ignoring the control group, two-way ANOVA was used to analyze the effect of two main factors (different silanes and treatments). Multiple comparisons were performed with Tukey tests (α=0.05).The dislodged posts were examined under a stereomicroscope  at ×20 to determine the failure modes as follows: adhesive failure at the cement‒dentin interface, adhesive failure at the cement‒post interface, mixed failure as a combination of adhesive failures along with cohesive failure within the resin cement (the separated cement). The representative specimens of the dislodged posts were observed under a scanning electron microscope .Pull-out bond strength (PBS) mean values and standard deviations (in N) for the nine study groups are presented in p≤0.01).Considering the control group, the results of one-way ANOVA and post hoc Turkey tests revealed a significantly lower PBS in the control group (no silane) compared to those in the nine other groups (p<0.001). The interaction between the two factors was not significant (p=0.15). The effect of air-drying was significantly lower than that of warm air-drying and laser treatment (p<0.001); the two latter did not differ (p>0.05). The effect of Porcelain Primer was significantly higher compared to those of universal adhesive (p<0.001) and Bis-Silane (p=0.01), with a similar result for the two latter.Two-way ANOVA indicated that PBS was influenced by silane type and post-silanization procedures  Porcelain Primer was significantly more effective in enhancing retentive strength of the posts cemented in root canals than Bis-Silane and one-bottle universal adhesive containing silane.2) Warm air-drying of silanized post considerably improved the efficacy of the silanes tested. Alternatively CO2 laser treatment might be effective when a blow drier is not accessible in the dental clinic.


In vivo experiments were performed after an intraperitoneal injection of 25 mg/kg/day ACR of female BALB/c mice for 7 days. The majority of in vitro ACR-treated oocytes reached the metaphase-II stage following 18 hours of incubation, which was not significantly different from the control group. Maturation of the oocytes derived from in vivo ACR-treated mice was impaired significantly. Oocytes, reaching the M-II stage in the in vivo ACR-treated group, were characterized by a decrease in meiotic spindle mass and an increase in chromosomal disruption. In vitro GLY treatment resulted in the degeneration of all oocytes, indicating that ACR toxicity on female germ cells may occur through its metabolite, GLY. Thus, ACR exposure must be considered, together with its metabolite GLY, when female fertility is concerned.High acrylamide (ACR) content in heat-processed carbohydrate-rich foods, as well as roasted products such as coffee, almonds etc., has been found to be as a risk factor for carcinogenicity and genotoxicity by The World Health Organization. Glycidamide (GLY), the epoxide metabolite of ACR, is processed by the cytochrome P-450 enzyme system and has also been found to be a genotoxic agent. The aim of this study was to determine whether ACR and/or GLY have any detrimental effect on the meiotic cell division of oocytes. For this purpose, germinal vesicle-stage mouse oocytes were treated with 0, 100, 500, or 1000 μM ACR or 0, 25, or 250 μM GLY  Acrylamide (ACR) is a colorless and odorless industrial compound formed by the hydration of acrylonitrile. It is widely used in the treatment of wastewater, paper/pulp manufacturing, mining, scientific research, surface coatings, textile and cosmetics , 2. Enviet al. [et al. [et al. [et al. [ACR was first defined as a neurotoxic agent in 1989, after a detailed study on workers who were exposed to ACR and acrylonitrile for two or more years . It was et al.  discoveret al. . In Apri [et al.  publishe [et al. . In June [et al. . The rep [et al.  document [et al.  also detThe primary pathway responsible for the ACR genotoxicity is the production of GLY through CYP2E1 . As reviin vitro and in vivo, focusing on meiotic maturation.One possible mechanism of ACR-related neurotoxicity is the interference of GLY with kinesin-related motor proteins, which also play a crucial role in meiotic division . Both meAll reagents were purchased from Sigma-Aldrich  unless otherwise stated.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Republic of Turkey Ministry of Food, Agriculture and Livestock. Ethical approval of the study was obtained from Ankara University Local Ethical Board of Animal Experiments . All animals were housed in an appropriate temperature and dark-light cycles controlled room in the Laboratory Animal Unit of Ankara University. Mice were sacrificed by cervical dislocation.All statistical analyses were performed through an SPSS software package . A chi-square test was utilized to compare groups. The significance level was set at p < 0.05.2 at 37°C for 18 hours.The ovarian follicular development of 21-day-old female BALB/c mice (n = 37) was stimulated by the intraperitoneal injection of 0.5 IU pregnant mare’s serum gonadotropin. Animals were sacrificed 48 hours following injection, and oocytes were collected from the ovaries by follicular puncture. Denuded GV-stage oocytes (n = 1084) were transferred into gamete maturation medium  and incubated in a humidified atmosphere of 5% COin vitro and in vivo effects of ACR on oocyte maturation were tested by two different sets of experiments. To test the in vitro effects of ACR on oocyte meiotic maturation, GV-stage oocytes (n = 452) collected from female BALB/c mice (n = 15) were cultured in gamete maturation medium containing progressive doses of ACR  for 18 hours. M-II stage oocytes were then fixed with a microtubule-stabilizing buffer containing 2% formaldehyde and 0.1% Triton-X for 30 minutes at 37°C to assess the meiotic spindle and chromosome morphology.ACR  was dissolved in ultrapure water to prepare a 10 mM stock solution. Working solutions were freshly prepared and dissolved either in a culture medium or 0.9% physiological saline depending on the experimental procedure. The in vivo effects of ACR were tested by the intraperitoneal injection of ACR to female BALB/c mice (n = 12) for 7 days (25 mg/kg/day). Oocytes were then collected from the ovaries as described above. The maturation stage of the oocytes (n = 342) was determined with an inverted microscope  equipped with a heating stage. M-II stage oocytes were fixed as described above.The in vivo effect of ACR was related to GLY metabolism or not, GV-stage oocytes from female BALB/c mice (n = 10) were exposed to 0, 25 or 250 μM GLY in vitro. One millimolar stock GLY  solution was prepared by dissolving 87.08 mg GLY in 1 mL ultrapure water. Working solutions were freshly prepared and dissolved in a culture medium. The maturation of oocytes (n = 290) was monitored for 18 hours and the maturation stage of each oocyte was determined.To determine whether the Fixed oocytes were incubated in a 1:100 dilution of anti-α-tubulin/anti-β-tubulin (1:1 mixture) mouse monoclonal antibodies for 90 minutes at 37°C for the evaluation of meiotic spindle integrity. Oocytes were then washed in phosphate-buffered saline (PBS) and incubated in a 1:100 dilution of an affinity-purified fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG  for 60 minutes at 37°C. Oocytes were stained with 10 μM 7-aminoactinomycine-D (7-AAD) as a nuclear stain. Finally, oocytes were mounted between glass slides and coverslips with a glycerol/PBS solution (1:1 mixture) containing 25 mg/mL sodium azide as an anti-fading reagent.Slides were assessed with a laser scanning confocal microscope  equipped with 488 nm Argon, 543 nm He-Ne and 633 nm He-Ne lasers and a 63× Zeiss Plan-Apo objective. Single and z-axis optical sections were collected, and 3D images were constructed using LSM-510 software.in vitro maturation was monitored following treatment to GV-stage oocytes with 100, 500 and 1000 μM ACR. Following 18 hours of incubation, 55.2% of the oocytes reached the M-II stage in the control group. There was no significant difference between the control and ACR-treated groups in terms of meiotic resumption of the oocytes  (Meiotic spindle formation during = 0.373) . The mei= 0.373)  and ACR-= 0.373)  groups. = 0.373) .Isolated GV-stage oocytes from the ovaries of control or ACR-treated mice were incubated in a maturation medium for 18 hours. Oocytes were then subjected to meiotic resumption analyses. The percentage of M-II stage oocytes in the control group (55.2%) was significantly higher than in the ACR-treated group  . Althougin vivo and in vitro ACR experiments led us to focus on the effects of GLY, the epoxide metabolite of ACR. To this end, isolated GV-stage oocytes were treated with 25 or 250 μM GLY for 18 hours in vitro. All oocytes were degenerated in both GLY treated groups, whereas 55.2% of the oocytes reached the M-II stage in the control group  .in vitro maturation of GV-stage oocytes was monitored following in vitro ACR treatment. Our findings showed that 100, 500 or 1000 μM ACR exposure did not block the in vitro maturation of mouse oocytes. In contrast, a recent study revealed that even low doses (10 and 20 μM) of in vitro ACR exposure to cumulus–oocyte complexes may cause a significant decrease in the number of mature oocytes. In the same study, chromosome misalignment and abnormal spindle configurations were distinguished following in vitro ACR exposure [in vitro ACR treatment had no adverse effects on meiotic spindle or chromosomes. Since Liu et al tested the in vitro effects of ACR on cumulus-enclosed oocytes, we wanted to examine the effects of ACR on denuded oocytes. As discussed later in this section, we hypothesize that the incubation of oocytes with the granulosa cells may have caused this effect.According to our previous experience, particular environmental agents such as dicoumarol , 25 and exposure . On the in vivo ACR-treated (25 mg/kg/day) BALB/c mice were subjected to meiotic resumption analysis. Our findings demonstrated that in vivo ACR treatment impaired oocyte maturation. The number of M-II stage oocytes was significantly higher in the control group compared to the ACR-treated groups. Moreover, a diminished meiotic spindle mass and disrupted chromosome alignment were observed in M-II stage oocytes following in vivo ACR exposure. Likewise, Duan et al. [et al. [in vivo ACR exposure caused a decrease in the number of healthy follicles and an increase in the apoptotic cells. Hulas-Stasiak et al. also argued that ACR exposure breaks oocyte–cumulus cell connections by disrupting vimentin filaments resulting in apoptosis [et al. [et al. [Secondly, oocytes derived from n et al.  noted thn et al. . In the  [et al. , the ovapoptosis . Similar [et al.  reported [et al.  evaluatein vivo and in vitro ACR experiments in our study led us to focus on the effects of GLY, the epoxide metabolite of ACR, which was previously suspected to cause genotoxicity [et al. [et al. [Inconsistency between the results of toxicity , 31. Sic [et al.  suggeste [et al. , 32. Con [et al. . It has  [et al. . In addi [et al.  determinin vitro. GLY exposure resulted in the degeneration of all oocytes, indicating that ACR toxicity may occur through GLY metabolism. It is known that the conversion of ACR to GLY is catalyzed by the CYP2E1 enzyme, a member of cytochrome P-450 family [in vitro oocyte maturation process in cumulus-enclosed oocytes but not in denuded oocytes. Therefore, the existence of the CYP2E1 enzyme in either granulosa cells or oocytes appears to be important. According to a study by Sobinoff et al. [et al. [et al. [in vivo and in vitro ACR experiments are consistent with the findings of Ghanayem et al. [et al. [in vitro ACR results are compared to our study, in which only denuded oocytes were studied.To test whether GLY is involved in ACR toxicity on female germ cells, isolated GV-stage oocytes were treated directly with 25 or 250 μM GLY for 18 hours 0 family . One of f et al. , granulo [et al. , CYP2E1- [et al. , indicat [et al.  also repm et al.  Although [et al.  conflictIn conclusion, since the direct exposure to ACR does not have toxic effects on mouse oocytes whereas systemic exposure does, we hypothesize that ACR toxicity is caused by its metabolite GLY and we believe that future studies focusing on ACR metabolites, particularly focusing on microtubule polymerization dynamics and microtubule related proteins, should be performed. In one study, internal doses of ACR and GLY were evaluated in humans. Tolerable daily intake (TDI) for neurotoxicity for ACR was estimated to be 40 μg/kg-day whereas the TDI for cancer was estimated to be 2.6 and 16 μg/kg/day for ACR and GLY levels, respectively . A dose–


P300-based BCI is one of the most common used BCI systems that can obtain high classification accuracy and information transfer rate (ITR). Face stimuli can result in large event-related potentials and improve the performance of P300-based BCI. However, previous studies on face stimuli focused mainly on the effect of various face types  on the BCI performance. Studies on the influence of face transparency differences are scarce. Therefore, we investigated the effect of semitransparent face pattern (STF-P) (the subject could see the target character when the stimuli were flashed) and traditional face pattern (F-P) (the subject could not see the target character when the stimuli were flashed) on the BCI performance from the transparency perspective. Results showed that STF-P obtained significantly higher classification accuracy and ITR than those of F-P ( Brain-computer interface (BCI) is a technology that allow users to communicate with others or control external devices via brain activity alone –3. BCI dP300-based BCI is one of the most common used BCI systems presented by Farwell and Donchin , and thiTen healthy subjects  participated in this study. The native language of the subjects is Mandarin Chinese. In addition, these subjects are all right-handed and have no known neurological disorders. They signed written consent form prior to the experiment, and the local ethics committee approved the consent form and experimental procedure before any subjects participated. Furthermore, all subjects had no previous BCI experiences.C = n!/(k!(n − k)!), 0 ≤ k ≤ n, where n represents the number of flashes per trial and k represents the number of flashes per trial for each element in the matrix. We chose the combination of C to denote the 12-flash pattern. The subjects sat in a chair in front of the monitor, which displayed a 6 × 6 matrix with characters and numbers  10]. Th. Th10]. Each subject was required to complete two paradigms (F-P and STF-P) on the same day. Each paradigm contained one offline block and one online block. The order of the paradigms F-P and STF-P was counterbalanced throughout the experiment (five of ten subjects did the F-P first). During the offline period, each paradigm consisted of three offline runs called one offline block, and each offline run contained 5 target characters that would be spelled by the subjects without any rest. The subjects had 3 min rest between each offline run. In addition, each target character was identified through 16 trials, and each trial was composed of 12 flashes. No feedback would be presented to the subjects in the offline experiment. However, during the online period, each paradigm only had one online run called one online block. The number of trials for recognizing each target character was selected automatically by an adaptive strategy , 18, andμV, band-pass filtered between 0.5 and 30 Hz, and sampled at 256 Hz. A total of 16 corresponding electrode positions were selected in the experiments according to the International 10-20 System  with a sensitivity of 100 0 System  8]. The. TheμV, Feature extraction is an effective method for reducing dimensionality and amount of required computations . In thisBayesian linear discriminant analysis (BLDA) was used to build the classifier model for the online experiment. BLDA can effectively solve the problems of high-dimensional data sets or noise fitting owing to its regularization method. Hoffmann et al.  applied The system was used to judge whether the results of two consecutive outputs were consistent. Accordingly, the final results of system output could be determined. If the results of two consecutive outputs were consistent, then the system exported the result as a feedback. Otherwise, the system would not provide any response until 16 trials were completed. When 16 trials ended, the classifier would automatically select the last output .t-tests (one-sample Kolmogorov-Smirnov test) for normal distribution to investigate the differences in mean amplitudes averaged from each ERP peak point ±20 ms between the F-P and STF-P paradigms. We also used such a method to explore the differences in online classification accuracy and bit rate between the two paradigms. The nonparametric Wilcoxon signed-rank test was used to compare the responses from the report of the subjects, as these data obey an uncertain distribution. The alpha level was α = 0.05.We chose paired-samples Did this paradigm make you tired?Was this paradigm difficult?Did this paradigm make you annoyed?After finishing the tasks of two paradigms, we conducted a questionnaire survey of three questions. The three questions were answered by the subjects on a 1–3 scale. A high score indicated a high degree of tiredness, difficulty, and annoyance . The questions were as follows:t = 2.49, p < 0.05, df = 9, t = 0.35, p > 0.05, df = 9 for VPP, and t = 1.45, p > 0.05, df = 9 for P300, Figures t = −0.65, p > 0.05, df = 9; t = −1.70, p > 0.05, df = 9; t = −2.69, p < 0.05, df = 9; R-squared values of ERPs from the two paradigms at 0–1000 ms averaged from subjects 1–10 on 16 electrodes. R-squared values of ERPs reflected the time energy of the signals. The definition is as follows:X1 and X2 are the features of classes “1” and “2,” respectively and N1 and N2 are the number of samples.t = 2.89, p < 0.05, df = 9 for classification accuracy, t = 4.03, p < 0.05, df = 9 for bit rate). Moreover, the number of trials for averaging of STF-P was significantly less than that of F-P .p > 0.05), degree of difficulty (p > 0.05), and degree of annoyance (p > 0.05).This study aimed to survey whether any difference would be found between STF-P in which the subject could see the target character during the time the stimuli were on and F-P in which the target character was concealed during the time the stimuli were on. The results showed that the STF-P could elicit larger N200 component and improve the classification accuracy and bit rate of the BCI system compared with the F-P.The Eriksen flanker task  is a comThe difference of P300 amplitude between two paradigms in this paper was not clear and no significant difference was found. This phenomenon may be attributed to the low luminosity contrast when the background is black . However, low luminosity contrast leads to low visual fatigue. Li et al.  studied The effects of repetition can decrease the amplitude of N400, especially for long-term offline data recording . In STF-p < 0.05). As in all, the STF-P could obtain superior performance compared to the F-P.Classification accuracy and ITR are the important indexes of BCI performance. In previous studies, face paradigms themselves have had good performances in accuracy (mean accuracy was higher than 90%) . TherefoThis research studied two paradigms only (semitransparency and nontransparency) and focused less on the different transparent degrees based on the state of being transparent. However, this research provided a new idea on the studies of face stimuli and demonstrated that other distinct components could contribute strongly to the BCI performance.In this study, we measured the performance of STF-P and F-P on BCI. The result indicated that STF-P was superior to F-P. In future studies, we will further verify the performance of the STF-P pattern on patients.


PKSs of this class produce the polyketide backbone of antibacterials (erythromycin), antifungals (amphotericin), immunosuppressive agents (rapamycin and FK506) and anticancer compounds (epothilone). In microbial genomes, PKS genes are often co-localized with all other genes required for the production of a given compound in a biosynthetic gene cluster. With the increasing ease of DNA sequencing, hundreds of novel assembly-line PKS gene clusters have been identified in recent years.3Polyketide synthase (PKS) enzymes catalyze the biosynthesis of polyketide natural products.4Escherichia coli is an attractive host for many reasons: it is easy to culture with a well-developed genetic toolbox, the primary metabolism is well understood, and because it is not an endogenous producer of polyketides, potential interference of native proteins with heterologously expressed PKS pathways may be limited.5 Despite these advantages, attempts at heterologous production of polyketides in E. coli have met with limited success. Examples of assembly-line PKS pathways expressed in E. coli include those encoding epothilone6 and 6-deoxyerythronolide B (6-dEB), the precursor to erythromycin.7 Titers for 6-dEB have ranged from 1 to 20 mg l−1, whereas titers for epothilone were significantly lower (0.001 mg l−1). These low titers have limited the utility of E. coli as a host for production of assembly-line polyketides, and have spurred efforts to improve general characteristics of this host to produce this class of compounds.7Heterologous expression of natural product genes in model hosts has been applied to access novel natural products including polyketides.E. coli as a host has been to increase availability of the substrates of PKS enzymes.8 For many assembly-line PKS pathways, the extender unit for the polyketide chain is (2S)-methylmalonyl-CoA (mmCoA), a metabolite not naturally produced by E. coli.9 Murli et al.10 compared three routes of heterologous mmCoA production in E. coli: (1) carboxylation of propionyl-CoA by the propionyl-CoA carboxylase (PCC) from Streptomyces coelicolor, (2) conversion of succinyl-CoA to (2R)-mmCoA and then to (2S)-mmCoA by the mmCoA mutase/epimerase from Propionibacterium shermanii and (3) synthesis of (2S)-mmCoA from methylmalonate by the malonyl/methylmalonyl-CoA ligase from S. coelicolor. The 6-dEB titers varied depending on the means of mmCoA production, with the PCC system consistently proving the most productive. These results suggested that, despite the low turnover rate of the 6-deoxyerythronolide B synthase (DEBS),11 the choice of pathway for heterologous mmCoA production has an effect on downstream polyketide titers. We hypothesized that further optimization of this upstream step, particularly of the promising PCC pathway, might further increase titers.These limitations point to a need for systematic optimization of heterologously expressed pathways. One approach to improving S)-mmCoA. In actinomycetes, such as S. coelicolor, the core catalytic PCC complex consists of α and β-subunits, each encoded by a distinct gene. The α-subunit consists of biotin carboxyl carrier protein and biotin carboxylase domains while the β-subunit has carboxyltransferase activity. Structural studies in S. coelicolor demonstrated that the β-subunits are homohexamers and implied an α6β6 PCC complex architecture.12PCC is a biotin-dependent enzyme that catalyzes the carboxylation of propionyl-CoA to 7 led to production of 6-dEB, the aglycone precursor of erythromycin. In this system, we observed poor expression of the S. coelicolor PCC α-subunit  sequence with the RBS sequence upstream of the PCC β-subunit, because the PCC β-subunit was highly expressed  pre-RBS sequence upstream of the newly substituted RBS. This method correctly predicted low expression of the wild-type PCC α-subunit. Combining these three changes resulted in an optimized construct . To facilitate standardized DNA synthesis and to assay protein expression for all PCC complex homologs, we made three major changes from the initial ‘wild-type’ design. First, each gene was flanked by an N-terminal His6 tag and a C-terminal Flag tag  on one plasmid  PCC identity affects DEBS protein expression. Measuring mmCoA concentrations and DEBS protein expression may clarify among these and other possible explanations.We generated strains of  plasmid , and the ref. −1  demonstrMyxococcus fulvus produced the highest titer of 6-dEB among the 13 strains tested, despite low levels of PCC protein expression (S. coelicolor (the wild-type version of which had been previously studied) produced the third-highest titer. Four PCC pathways have been previously characterized in terms of kinetic parameters, but there was no observable trend between Km and 6-dEB production (Table S5).The strain harboring a PCC homolog from pression . The strE. coli strains harboring the 13 engineered PCCs to a strain harboring the previously tested wild-type S. coelicolor PCC  than all 13 strains harboring engineered PCC homologous complexes, including that of the engineered S. coelicolor PCC (3 mg l−1) and even the best-performing engineered homolog from M. fulvus  epitope tags were added to the engineered PCC subunits, (2) the 5′UTR upstream of each PCC subunit was modified, and (3) synonymous mutations of the pcc genes were engineered to match E. coli codon usage.This large difference in titer between the wild-type 6β6 architecture in several organisms, including species of Streptomyces21 and Myxococcus,22 we first considered the possibility that epitope tags could influence the protein complex in terms of stability or activity. To test the hypothesis that epitope tags on PCC proteins reduced polyketide titers, we built several new strains harboring engineered S. coelicolor and M. fulvus PCC complexes with presence and absence of N- and C-terminal epitope tags (S. coelicolor (20 mg l−1 vs 3 mg l−1) and M. fulvus  complex, which exists natively in all bacteria, including oduction , it is pE. coli. We determined that variations in the PCC complex led to variation in polyketide production, suggesting that the PCC complex is, in some contexts, rate-limiting. This may be due to different activities of individual PCC enzymes when expressed in E. coli, differences in their solubility or biotinylation, or due to an effect on DEBS protein expression. Future work quantifying mmCoA and protein expression may determine the cause of the variability. We identified a PCC complex from the myxobacterium M. fulvus that outperformed all engineered PCC complexes in terms of polyketide production. We characterized the best-performing PCC homologs in S. coelicolor and M. fulvus, determining that the epitope tags drastically reduce polyketide production. This work demonstrates that, of the systems tested, the PCC complex from S. coelicolor supports the highest levels of heterologous polyketide production. The molar yield of 6-dEB of the E. coli strain, harboring the wild-type S. coelicolor PCC, which produced the highest 6-dEB titer, is 1.2%, which is in good agreement with what has been previously reported in literature.29 However, using flux balance analysis, the maximum theoretical molar yield of 6-dEB was calculated to be 10.7%.29 Given the poor-expression level and relatively high titer, it is possible that optimization of the M. fulvus PCC subunits, including eliminating all epitope tags, or by co-expressing additional open reading frames (In conclusion, we investigated the effect of expressing 13 heterologous PCC complexes from different species on polyketide production in g frames , could l


The objective of this study was to evaluate the influence of masticatory muscle injection of botulinum toxin type A (BTX-A) on the growth of the mandibular bone in vivo.n = 6) or saline (n = 5) was injected at 13 days of age. All injections were given to the right masseter muscle, and the BTX-A dose was 0.5 units. All of the rats were euthanized at 60 days of age. The skulls of the rats were separated and fixed with 10% formalin for micro-computed tomography (micro-CT) analysis.Eleven Sprague-Dawley rats were used, and BTX-A (P < 0.05), and the mandibular plane angle of the BTX-A-injected group was significantly greater than in the saline-injected group (P < 0.001). In the BTX-A-injected group, the ramus heights II and III and the mandibular plane angles I and II showed significant differences between the injected and non-injected sides (P < 0.05). The BTX-A-injected side of the mandible in the masseter group showed significantly lower mandibular bone growth compared with the non-injected side.The anthropometric analysis found that the ramus heights and bigonial widths of the BTX-A-injected group were significantly smaller than those of the saline-injected group (BTX-A injection into the masseter muscle influences mandibular bone growth. Clostridium botulinum [Botulinum toxin type A (BTX-A) is a bacterial neurotoxin produced by the gram-positive bacterium otulinum . BTX-A iotulinum . It blocotulinum . BTX-A wotulinum . BTX-A hotulinum , 6. BTX-otulinum , 7. BTX-otulinum .Mandibular bone morphology can be affected by the masticatory muscle function and activity . In funcBTX-A can be selectively administered in the masticatory muscles and can decrease muscle activity without surrounding tissue damage . In a prIn the present study, we evaluated how the BTX-A injection into the masseter muscles influenced the growth of the mandibular bone in a growing rat model. We measured the anthropometric points and linear distances in the craniofacial bones and compared the changes in bone structure. In this study, we hypothesized that BTX-A injection into the unilateral masseter muscle would induce lower growth rate and a deviated mandible. Thus, the purpose of this study was to evaluate the effects of muscle hypofunction due to BTX-A injection on mandibular bone growth.n = 6). The same amount of saline was injected into the right masseter muscle in the control group (n = 5). The administration of saline and BTX-A was performed at the same time in both groups. All the rats were euthanized at 60 days of age (47 days after injection). The skulls of the rats were separated and fixed with 10% formalin for micro-computed tomography (micro-CT) analysis. This study was approved by the Institutional Animal Care and Use Committee of the Gangneung-Wonju National University, Gangneung, Korea (IACUC GWNU-2016-24).Eleven Sprague-Dawley rats were used in this study. The rats were randomly divided into the control and the experimental groups. At 13 days of age, BTX-A or saline was injected into the masseter muscle. The BTX-A  was prepared and diluted with 50 mL of saline. BTX-A (0.5 unit) in a 0.5-mL dose was injected intramuscularly into the right masseter muscle in the experimental group /CT/single photon emission computed tomography (SPECT) system  at the Ochang Center at the Korea Basic Science Institute. The CT scanner was set to an 80 KV voltage for the X-ray tube, 500 μA current for the X-ray source, and 210 ms of exposure time. The detector and the X-ray source were rotated 360 degrees in 360 rotation steps. The number of calibration exposures was 30. The system magnification was allowed over 30.7 mm of the axial field of view (FOV) and 30.7 mm of the transaxial FOV. The scanned images were reconstructed with an Inveon Research Workplace Software . The calibrated three-dimensional (3D) images were shown in gross profiles of the skulls of the rats.Anthropometric points were identified, and the distance was measured for the evaluation of mandibular growth changes on micro-CT images. Anthropometric points are shown and described in Fig. t test was used. For the comparison of the control group and the experimental group, an independent sample t test was used. Differences with P values less than 0.05 were considered significant.When there were two compared groups and the comparisons were performed in the samples within the same animal, a paired P = 0.025 and 0.002, respectively). The average total mandibular and corpus lengths in the experimental group were less than in the control group (Table P = 0.008).Most of the average vertical anthropometric measurements of the mandibular bones in the experimental (BTX-A-injected) group were less than in the control  group Table , Fig. 3.P < 0.001). The dental midline of the lower anterior tooth compared with the upper tooth was deviated to the right side by 0.035 ± 0.118 mm in the experimental group.For the transverse measurements, only the bigonial mandibular width was significantly different between the saline-injected group and the BTX-A-injected group . The average sagittal measurements of mandibular plane angles I and II were greater on the right side (BTX-A injection) than on the left side. There were significant differences in the mandibular plane angles I and II .There was no significant difference between the right and left sides of mandibular bone growth in the saline-injected group Fig. . In the Mandibular bone growth can be affected by masticatory muscle function and activity . HyperacBTX-A could decrease muscle activity and show paralytic effects immediately after injection in a rabbit model . The maxBTX-A can be safely used in the mandibular region because only a small amount of BTX-A is required for the treatment of the muscle disorder or for cosmetic procedures, compared with spasticity treatment , 27. TheDecreased muscle activity can affect the bone metabolism and remodeling processes . The dimP < 0.05; Table BTX-A injection into the masseter muscles before bone development caused the low growth of the mandibular bone , 22, 23.The influence of paralytic effects of BTX-A on the mandibular bone growth is a clinically valuable result. BTX-A has been used for facial muscle disorders, TMJ problems, and cosmetic procedures . RecentlP = 0.025, 0.002, and 0.006) and increased mandibular plane angle II, compared with the saline-injected group (P < 0.001). Further, the BTX-A-injected side had significantly smaller ramus height and significantly higher mandibular plane angle, compared with those of the non-injected side (P < 0.05). From these results, we could confirm that BTX-A injection into the masseter muscle during the growth phase could cause low growth of the mandibular bone. This animal study was useful for evaluating the degree of the low skeletal bone growth effects of BTX-A. Anthropometric evaluations showed that BTX-A injection affected the bony growth pattern.In this study, we evaluated the paralytic effects of BTX-A on the mandibular bone growth in vivo. The injection of BTX-A into the masseter muscle was performed in 13-day-old rats. Anthropometric measurement analysis showed that the BTX-A-injected group had significantly decreased ramus heights I and III and bigonial mandibular width (


Arabidopsis thaliana accessions. A survey of accessions revealed that most exhibit a vigorous rapid-flowering response in a FR-enriched environment. However, a subset of accessions appeared to be compromised in the accelerated-flowering component of the shade-avoidance response. The genetic basis of the muted response to FR enrichment was studied in three accessions . For all three accessions, the reduced FR flowering-time effect mapped to the FLOWERING LOCUS T (FT) region, and the FT alleles from these accessions are expressed at a lower level in FR-enriched light compared to alleles from accessions that respond robustly to FR enrichment. In the Mir-0 accession, a second genomic region, which includes CONSTANTS (CO), also influenced flowering in FR-enriched conditions. We have demonstrated that variation in the degree of precocious flowering in shaded conditions (low R:FR ratio) results from allelic variation at FT.Plants have evolved developmental mechanisms to ensure reproduction when in sub-optimal local environments. The shade-avoidance syndrome is one such mechanism that causes plants to elongate and accelerate flowering. Plants sense shade via the decreased red:far-red (R:FR) ratio that occurs in shade. We explored natural variation in flowering behavior caused by a decrease in the R:FR ratio of  Plants have evolved the ability to alter morphology and the timing of flowering as an adaptation to changes in the local environment. One such mechanism is the shade-avoidance response, which allows plants to compete with neighboring vegetation for sunlight. When challenged with a shaded environment, plant stems and petioles elongate in an attempt to grow above the canopy and capture more light. Moreover, shaded plants often reproduce earlier than non-shaded plants to ensure that at least some limited seed set occurs in the suboptimal environment .Changes in light quality are perceived by the photoreceptors including PHYTOCHROMES A-E (PHYA-E), CRYPTOCHROMES 1 and 2, and PHOTOTROPINS 1 and 2 (for review see ). In shaGIGANTEA expression, and GIGANTEA is required for CONSTANS (CO) expression [CO expression oscillates during a 24 hour period, and stability of the CO protein is light dependent, with degradation occurring in the dark [CO promotes flowering by activating expression of downstream floral integration genes, such as FT and SUPPRESSOR OF CONSTANS 1, and FT expression is a key inducer of flowering [The circadian clock and phytochromes are part of the system that monitors day-length and mediates changes in the expression of gene networks. The circadian clock regulates pression . CO exprthe dark . CO promlowering . Thus, bFT was initially genetically identified by Koornneef et al. [FT gene, more recently called FLOWERING LOCUS T, was cloned in 1999 (11). FT is activated by CO in long-day photoperiods in long-day plants, such as Arabidopsis [FT is now recognized as “florigen,” a mobile signal that promotes flowering . The anntal cue –15.FT expression is an integration point at which multiple environmental signals allow plants to fine tune flowering time as a function of environmental conditions. In addition to photoperiod, FT expression is influenced by vernalization (extended cold), ambient growth temperature, and hormone levels [FT promoter that are likely to be key determinants for expression via binding of different proteins or protein complexes [FLC (FLOWERING LOCUS C), a potent floral repressor silenced by vernalization, as well as the repressor SHORT VEGETATIVE PHASE, bind directly to the FT promoter [CO and GA (gibberellic acid) responsive proteins activate expression of FT [FT it is likely to be a result of cis-regulatory element variation, because the FT amino acid sequence is quite highly conserved among Arabidopsis accessions that are highly variable for flowering time.The control of e levels –20. Receomplexes , 22. FLCpromoter , 24. CO on of FT , 25. If GI and CO [FT and is correlated with FT expression levels. For one group, FT is the only locus identified as causing FR insensitivity, whereas for a second group, both FT and CO are candidate genes.In this study, we evaluate natural variation for flowering time in FR-enriched conditions that mimic shade. The majority of Arabidopsis accessions flower rapidly in these conditions, with the exception of two groups of accessions, which appear to be “blind” to the increased FR light. Previous work has demonstrated that FR enrichment overrides the repressive effect of FLC and alters the timing of expression of I and CO –29. Hereco-9 [ft-3 [Accessions were obtained from Julian Maloof and Todd Michaels in 2002. Strains were described as follows: ColFRI , co-9 [3co-9 , ft-3 [1-9 [ft-3 , Est-1 N-9 [ft-3 .-2s-1) set for long days  or short days . All experiments were conducted at 22C, except for the cycling experiments in which the 16h light period was at 22C and the 8h night period was at 16C. Certain chamber shelves also contained arrays of FR LEDs  that lowered the R:FR ratio from 5 to 0.15 (low FR) or 0.04 (high FR). Vernalization treatment was done in soil in a coldroom in the dark at 4C. Plants grown outdoors were first grown in growth chambers for 3 weeks and then moved outside to a partially shaded (70%) location in Madison WI beginning in early June. All light measurements were made with a wideband portable spectroradiometer .All growth-chamber experiments were conducted in reach-in chambers  fitted with T12 fluorescent bulbs  on the primary shoot prior to flowering, with the exception of plants grown outdoors where Days to Flowering (DTF) was used due to excessive growth of secondary shoots. Genotypes were determined by SSLP or CAPS markers using TaKaRa ExTaq RR001A.http://www.rqtl.org) in the statistical package R (https://www.r-project.org).Correlation coefficients were calculated using Microsoft Excel (CORREL). QTL mapping utilized R/qtl . Similarily, FT expression in the other heterozygous accessions was monitored and digested with Cac8I  using primer set (5’-CAATCAACACAGAGAAACCACCTG-3’ and 5’- CTCGCGAGTGTTGAAGTTCTG -3’).To monitor enerated . We usedOur studies of late-flowering, vernalization-insensitive accessions revealed that most of these accessions flower relatively rapidly when exposed to FR-enriched light. However, a small subset does not exhibit accelerated flowering with FR enrichment. To characterize the FR-flowering phenotype and investigate how it relates to other known pathways that accelerate flowering, we grew 36 accessions under 5 conditions, including FR enrichment. The accessions .To further characterize the FR phenotype, we characterized another set of 36 accessions , were chosen for further study and grown in 10 different conditions . Fig 3 sFT.To determine the genetic basis of the FR-flowering time differences, crosses were performed among the 10 accessions and controls with different FR-flowering behaviors. F1 plants grown +/- FR show that FR responsiveness is largely dominant with a small dosage dependency effect since most F1 plants have a flowering time nearer the early/FR sensitive parent . Fig 4 sFT in FR sensitivity was provided by a second population, generated by crossing a flowering time FR-insensitive accession (Fl-1) to ColFRI, in which the FR phenotype in the F2 generation segregated in 3:1 manner with early flowering being dominant (FT (F5I14). The results demonstrate the strong effect of the Fl-1 FT region in delaying flowering under FR enrichment . In addition, FLC was genotyped in this population and showed no correlation to flowering time (data not shown).Further evidence for the role of dominant . Twenty FT. For some crosses, multiple independent F2 populations were analyzed, and the results were reproducible (data not shown). As already discussed, the association of the FT region and FR-enriched flowering in the Hau/ColFRI and the Fl-1/ColFRI F2 populations is strongly supported, whereas the FLC/CO region shows no support. In addition, for crosses between Sue-0 (semi-sensitive) and Et-0 (semi-sensitive) to ColFRI, flowering time was linked in the F2 to the FT region . However, one population, Mir-0/ColFRI, has relatively lower support at the FT region and significant support for the FLC/CO region in comparison to the other F2 populations, indicating natural variability in which locus/loci confers FR sensitivity (this population is discussed in more detail below). In addition, an F2 population from a FR insensitive accession (Mir-0) and a FR semi-sensitive accession (Sue-0) had significant linkage between FR-enhanced flowering and the FT region (p = 0.02), as would be expected. Likewise, when Fl-1 (insensitive) was crossed to Hau-0 (semi-sensitive), the F2 FR flowering phenotype was dependent on the FT region (p = 0.01).Evaluation of F2 populations derived from crosses between FR semi-sensitive and insensitive strains to ColFRI demonstrated tight linkage of FR sensitivity to the region of chromosome 1 containing FT region, and QTL mapping revealed a QTL on chromosome one over the FT region LOD = 5.8, LOD >4.2 are significant). For the second F2 population, Mir-0/ColFRI, both chromosome 1  and chromosome 5  had significant support for having an effect on flowering time.We utilized a whole-genome scan  (149 SNPFT marker (F5I14). The earliest plants were enriched for ColFRI alleles (17/20) and the latest plants were enriched for Mir-0 alleles (15/20). Heterzygous plants were present in both the earliest and latest classes, suggestive of an additional causative locus. Using progeny lines fixed for FT (ColFRI) and FLC (ColFRI), but heterozygous for CO we observed a differential response to FR flowering attributable to the CO region . Thus, the almost complete FR insensitivity of Mir-0 is likely to be caused by natural variation at both FT and CO. A second accession, Br-0, was also different from the other FR insensitive accession, and displayed identical flowering behavior as Mir-0. Both accessions are also glabrous, suggesting possible relatedness. Thus Br-0 was included in the co and ft mutant analysis described below.The Mir-0 accession displays some unique behavior compared to the other FR-insensitive accessions. First, it is not extremely late flowering without FR (50 RLN), and flowers with approximately the same number of leaves as with or without FR enrichment when grown side by side. Second, Mir-0 is extra sensitive to vernalization, with even short cold treatments causing a reduction in flowering time. To pursue in more detail the behavior of this accession, we grew 96 F2 plants of Mir-0/ColFRI in FR-enriched conditions. The earliest and latest 10 plants were genotyped at a tightly linked FT in FR sensitivity, we evaluated flowering time in an F2 population from a Col ft mutant crossed to ColFRI , whereas one functional copy of FT was sufficient for FR sensitivity and relatively early flowering (<28 RLN). We also evaluated the co mutant in the ColFRI background and it also flowered extremely late in FR-enriched conditions, nearly identical to the ft mutant in the same genetic background (data not shown).Given the compelling data for a role of o ColFRI , and theFT and CO in FR responsiveness, we crossed multiple accessions to ft or co mutants in the ColFRI background and phenotyped the F1 plants for FR sensitivity  flowered substantially later (20+ RLN) than the parental accession, demonstrating an inability of the Mir-0 and Br-0 FT alleles to complement the FT mutant. In addition, when Br-0 and Mir-0 were crossed to a CO mutant, the F1 plants were equally late, comparable to the crosses with ft, showing a lack of co complementation. The mutant analysis combined with the previously described experiments support the idea that Mir-0 and Br-0 likely have alleles of CO and FT which do not respond to FR enrichment.To further our understanding of the role of sitivity . It is iFT appears to be critical for FR sensitivity. The semi-sensitive accession (Hau-0) crossed to ColFRI(ft-1) had an F1 FR-flowering phenotype equivalent to Hau-0. This demonstrates that the FT allele in Hau-0 retains some FR-sensitive FT function; however, the F1 from Hau-0/ColFRI(ft-1) is later than ColFRI, indicating that the FT allele from Hau-0 is less capable of responding to FR compared to the FT allele from ColFRI  had a flowering phenotype equivalent to the FR-sensitive accession  likely due to the dosage-dependent behavior of CO, and suggesting a functional CO in these accessions. Et-0 (semi-sensitive) crossed to FR-sensitive (sensitive) accessions (HOG and Nok-3) had flowering times equivalent to the FR-sensitive accessions, suggesting that the Et-0 CO allele is capable of acting on the FT alleles from those two accessions. In addition, the Et-0/ColFRI F2 showed no linkage at chromosome 5, demonstrating CO functionality in this accession . The Fl-1/ColFRI(ft-1) F1 population flowers as late as Fl-1 and ColFRI(ft-1), demonstrating that the Fl-1 FT allele has an altered function. Fl-1/ColFRI(co) F1 flowering time data (54 RLN) shows that the Fl-1 CO allele appears functional and is capable of acting on the ColFRI FT allele, further supporting the notion that Fl-1 FR insensitivity of Fl-1 results exclusively from an altered functional FT allele. Therefore, the data support the notion that FT allelic variability in natural accessions is a major factor in reduced FR sensitivity for flowering time.The only accession with an amino acid substitution in FT allele, which mapped to the FT promoter region [FT region in a Col background was crossed to ColFRI to determine if this allele of FT was compromised in FR-dependent flowering in a winter-annual (FRI+) background. The FR-flowering phenotype of the F2 population was dependent on the FT region with plants containing Col FT (14 RLN) flowering substantially earlier than plants containing Est-1 FT (38.6 RLN), providing additional evidence that FT plays a central role in FR-induced flowering.Using a Recombinant Inbred Lines (RIL) population, the Est-1 accession was shown to have a hypo-active r region . A NIL . After 5 weeks of FR treatment, all accessions/strains were expressing FT with ColFRI and Hau-0 having relatively high and equivalent expression, which correlates well with the FR responsiveness. Both Mir-0 and Fl-1 had not flowered after 5 weeks of FR and this correlates with their lower level of FT expression.We evaluated , +/- FR . WithoutFT alleles would be expressed at different levels, we evaluated F3 plants that are heterozygous at FT from 3 populations for allele-specific expression . It is tempting to speculate that this behavior is not just a consequence of population structure, but provides an adaptive advantage to accessions at higher latitudes that experience a long winter. One possibility is that because northern latitudes experience longer durations of twilight which is enriched in FR, insensitivity to FR could prevent flowering at inappropriate times of the growing season , 38. AnoCO and FT allelic variation is likely to contribute to insensitivity to FR-induced flowering. Why FR insensitivity is present in accessions from these two regions is unknown, but the relatively short vernalization requirement shared by these two accessions may render the lack of a FR response ecologically relevant, providing an additional layer of protection to prevent precocious flowering.Two accessions from southern Europe, Mir-0  and Br-0 , were also FR insensitive for flowering. Both accessions are glabrous, have identical flowering behavior, and were collected 378 miles apart, indicating the possibility of relatedness. These two accessions are also unique in that both FT expression is intimately associated with FR-induced flowering. In Arabidopsis, FT is one of the most highly conserved proteins with only a single amino acid variation across the species; thus, variability associated with FT must reside in expression of the FT gene, as demonstrated in this study. FT has both negative regulators  as well as positive regulators , all of which bind to the promoter region or the first intron [FT that could serve as binding sites for regulatory proteins that control FT transcription [FT expression. In addition to regulators and promoter motifs, a role for chromatin modification has been demonstrated providing an additional layer of regulation [FT, with no ORFs 7.5kb upstream and 9.5kb downstream, may be related to regulation by higher-order chromatin structure. Thus, FT regulation is complex as might be expected for an integrator that receives signals from multiple pathways . We have shown that FR enrichments leads to accelerated flowering in some accessions via variability in FT expression. FT, or florigen, is central to environmentally induced flowering and an attractive candidate for engineering plants for a changing environment.In summary, we demonstrate that t intron , 39, 40.cription , 41. Futgulation . The unuS1 FigGA accelerated flowering for all accessions except Fl-1. Fl-1 grew extensive secondary meristems, making scoring RLN inaccurate, thus the SD experiment was terminated after 60 days.(TIF)Click here for additional data file.S2 FigF1 populations had flowering times that mirror the flowering time of the most FR sensitive parent demonstrating dominance, and no F1 exceeded the flowering time of the less sensitive parent.(TIF)Click here for additional data file.S3 FigThe F2 population from a cross between Fl-1 (In) and ColFRI (Se) segregated in a 3:1 manner, suggesting one major-effect loci as conferring the phenotype in this population.(TIF)Click here for additional data file.S1 TableArabidopsis accessions used in this study, along with controls (Col and ColFRI), under various growth conditions.Flowering time data for (XLSX)Click here for additional data file.S2 TableTen accessions, representing much of the breadth in flowering time behavior, were phenotyped under 10 various growth conditions.(XLSX)Click here for additional data file.S3 TableCorrelations among the ten conditions for the 10 accessions from SD (XLSX)Click here for additional data file.S4 TableResults for accessions contained within this study and those used my Maloof, et. al, show no correlation between FR responsiveness for the two FR dependent phenotypes.(XLSX)Click here for additional data file.S5 TableF1 plants flower most like the early flowering (FR-sensitive) accessions, demonstrating dominance.(XLSX)Click here for additional data file.S6 TableOver 100 accessions were scored for multiple polymorphisms around FT, with two being within the ORF of FT, which was used for the allele-specific expression experiments (BsmBI and Cac8I). Only two accessions (Fl-1 and Bsch-2) contained the polymorphism responsible for the BsmBI site. In addition, the table shows data for an indel upstream of FT . The 1.2kb version is relatively rare being present in only 6 accessions, including two (Et-0 and Br-0), accessions used extensively in this study.(XLSX)Click here for additional data file.


Adult-type hypolactasia (ATH) is a clinical syndrome of primary lactase deficiency. A lactose-free diet is advisable to avoid the symptoms linked to the condition, but this potentially creates problems for optimal bone mineralization due to reduced calcium intake. To evaluate the effect of the lactose-free diet on the bone mineral status (BMS), we compared the phalangeal BMS of adolescents with ATH to that of peers on a normal diet. Also, we analyzed the correlations between BMS and dietary behavior, physical exercise, and calcium and vitamin D intake. A total of 102 cases and 102 healthy controls filled out a diet record and underwent phalangeal Quantitative Ultrasound (QUS). No difference in BMS was observed. The time spent on lactose-free diet (4.8 ± 3.1 years) was inversely correlated to the BMS. More than 98% of cases consumed lactose-free milk, but calcium and vitamin D intake were significantly lower. Calcium intake was correlated to physical exercise but not to BMS. Our results suggest that a lactose-free diet does not affect the phalangeal BMS of adolescents with primary lactase deficiency when their diet includes lactose-free cow’s milk. However, there is still a significantly lower calcium intake than in the population reference. The inverse correlation observed between the BMS and the time spent on a lactose-free diet suggests that a long-term follow-up is advisable. The ability to digest lactose depends on the gene coding for lactase , an enzyme located in the brush border of the small intestine and localized to the tip of the villi . LactaseSome studies reported that a reduced value of a quantitative ultrasound (QUS) variable, both velocity- and attenuation-based, was associated with a reduced BMS in children with growth problems or disorders affecting bone health ,12,13,14The aim of this study was to evaluate the effect of a lactose-free diet on the BMS in primary-lactase-deficiency adolescents and compare it to a similar population on a diet without restriction (regular diet) using phalangeal QUS, a radiation-free technique. Furthermore, we looked for possible correlations between phalangeal BMS and diet, physical exercise, and Ca and vitamin D intake.n = 41) or lactose-tolerant  on the basis of whether or not they had experienced gastrointestinal symptoms during the breath test.The case group  was formed by 102 northeastern Italian adolescents (age 16.8 ± 2.6 years) of Caucasian ancestry, who had been in the past referred to the pediatric Gastroenterology clinic of the University Hospital S. Anna, Ferrara, Italy (Latitude 44°50′0″ N). All of them presented a positive hydrogen breath test and had a genotype compatible with primary lactase deficiency. The cases were further subdivided in lactose-intolerant , matching the case group for age and sex, by means of a mathematical random function. In both groups, exclusion criteria were celiac disease, cystic fibrosis, or inflammatory bowel disease. In the control group, individuals who had LI or reported discomfort after ingesting dairy products were also excluded.2). All subjects were defined as underweight (under 3rd percentile), normal weight (between 3rd and 85th percentile), overweight (between 85th and 95th percentile), or obese (≥95th percentile) on the basis of the Cacciari’s growth chart percentiles for Italian children and adolescents [Both cases and controls underwent a physical examination with anthropometric evaluation and QUS measurement of the bone status. All cases and controls had reached the Tanner pubertal stage 5. A team formed by two pediatricians, one biostatistician, one dietitian, and one medical student went to the school, presented the research project, and asked the students to fill out a dietary record for the week preceding the day of the examination. The QUS apparatus was then taken to the school gymnasium to examine the volunteers. The anthropometric measures were obtained in the morning using a repeated-measure protocol . Height lescents . The stuwww.scottibassani.it). Bottled water was classified in three categories depending on the total dissolved solids (TDS) value reported on the bottle label: very low mineral content (TDS < 50 mg/L), low mineral content (50 mg/L < TDS < 500 mg/L), medium mineral content (500 mg/L <TDS < 1500 mg/L). Ferrara’s tap water TDS was provided by the public water company . The diet record required also information on the weekly hours of physical activity performed: if it was limited to the two hours at school or if the subject practiced extra-curricular activity; how many hours per week were devoted to the extra-activity; the type of exercise, i.e., weight-bearing  or non-weight-bearing . The Population Reference Intake (PRI) of nutrients for the Italian population (LARN) was based on the guidelines issued by the Italian Society of Human Nutrition (www.sinu.it) [The diet record was designed to analyze the diet during one week (7 days) to estimate, in particular, the average daily intake of Ca and vitamin D. The diet record considered consumption (amount and servings/day) of foods that are a source of Ca and vitamin D: milk and dairy products , water, vegetables, fresh and dry fruit, fish, and sweets and desserts containing dairy products. To define the quantity, the record contained pictures with example of servings from the dietary atlas by Istituto Scotti Bassani, Italy (sinu.it) . At the Quantitative ultrasound measurements were obtained using a DBM Sonic Bone Profiler BP01 . Ultrasound attenuation of the amplitude-dependent speed of sound (AD-SOS) and bone transmission time (BTT) were measured at the diaphysis of the proximal phalanx of the four fingers of the non-dominant hand ,21,22. IThe breath test had been performed for the cases for diagnostic purposes, as previously reported . In detat-test was used to analyze differences in quantitative data, and the values were given as mean ± standard deviation. The Shapiro–Wilk test was applied to check for normal distribution, and the Bartlett test to check for equal variances across the samples. When not normally distributed, the values were transformed using the Box–Cox transformation. If it was not possible to normalize the data, they were transformed as close as possible to a normal distribution. The Fisher’s exact test was used to examine the significance of the association in 2 × 2 contingency tables. Generalized linear models were performed to analyze the association of linear or binary outcomes with multiple variables and to adjust for covariates. The Pearson’s correlation coefficient (R) was computed for the parametric estimates of the level of association between the variables. For non-parametric variables, the Spearman’s correlation was used. A statistical analysis was performed with the software R version 3.2.2 . The following R packages were used: ‘irr’ version 0.84 to calculate the ICC, ‘AID’ version 1.5 for the Box–Cox transformation, and ‘pwr’ version 1.1.1 to check the statistical power based on the sample size, standard deviation, and mean to have type II error (beta) > 80%. A p-value < 0.05  was considered statistically significant.The Student’s 2 vs. 21.6 ± 3.8 kg/m2, p = 0.01) and weight  than males in the case group. No significant difference was observed between female cases and controls . When stratifying by sex, no statistical difference was observed between cases : 0.27 ± 1.35 vs. Male (M): 0.21 ± 1.40, p = 0.30) and controls .The AD-SOS age-related Z-score did not differ between cases and controls  both in cases and controls , also when adjusted for hours spent exercising and Ca intake. Twelve cases (11.8%) had low BMS. In the control group, five subjects (4.9%) had low BMS. The BTT age-related Z-score did not differ between cases and controls (p = 0.75). When stratifying by sex, no statistical difference was observed between cases  and controls .The AD-SOS age-related Z-score was inversely correlated to the BMI (p = 0.11) and BTT age-related Z-score (p = 0.07). No difference was found between males and females, both in cases and controls, when comparing the AD-SOS and BTT age-related Z-score.Among the cases, there was no difference between LI and LT subjects in terms of AD-SOS age-related Z-score . To further investigate, the cases who had been for more than 4.8 years on a lactose-free diet  were compared to a randomized control subgroup matched for number and age. There was no difference in the AD-SOS age-related Z-score (p = 0.13), while there was a difference in Ca intake .At the time of this study, the cases had been on a lactose-free diet for 4.8 ± 3.1 years. The AD-SOS age-related Z-score was inversely correlated to the time spent in the lactose-free diet (p = 6 × 10−4). This difference was not observed in the case group (p = 0.86).The lactose malabsorbers had a lower Ca intake and a lower Vitamin D intake than the control group . Controlp = 0.03)  , with nop = 0.62).There was no difference between the two subgroups in terms of Vitamin D intake (p = 0.78). In the control group, males introduced significantly more Ca than females (p = 0.001)About 75% of cases drank milk (98% of whom drank lactose-free cow’s milk and about 2% soy milk), and nearly 83% of controls drank cow’s milk with the same average frequency . There wLess Ca was significantly introduced by the cases through dairy products , fruit (fresh and dry), legumes, fish, and sweets and desserts containing dairy products. Parmesan cheese was considered separately from the aged cheese group because of its high Ca (1159 mg Ca/100 gr) and very low lactose content . The casWater provided 8.0% of Ca in the cases and 5.5% in the controls, the highest percentage deriving from water with medium mineral content  . There wp = 1 × 10−4, respectively). About 73% of the controls and 23% of the cases  did extra exercise in addition to the curricular hours at school. During the extra hours, about 98% of the controls and 72% of the cases practiced weight-bearing sports, mostly soccer and basketball. On average, the number of weekly hours spent exercising were 5.2 ± 3.2. The controls spent significantly more time practicing physical activity than the cases . There was no significant correlation between Ca intake and BMI (p = 0.44), either in the cases or in the controls. The number of weekly hours spent doing physical exercise did not correlate either with the AD-SOS Z-score (p = 0.16) or with the BTT Z-score (p = 0.30).A linear correlation was observed between the average Ca intake (mg/day) and the weekly hours spent doing physical exercise (p = 0.38) or BTT age-related Z-score (p = 0.21). In subjects with low BMS, there was no significant difference between cases and controls in Ca intake  nor in hours spent exercising . Cases and controls with low BMS (n = 17) did not significantly differ in Ca intake from those with normal BMS (n = 187) .No significant association was observed between dietary Ca intake and AD-SOS age-related Z-score , that may facilitate bone growth . LactoseIn addition to milk, water may be another important source of Ca, as the dissolved minerals are highly bioavailable . In our The data showed that Ca intake, as well as the intake of vegetables, legumes, fruit and fish in children and adolescents was lower than recommended, both in cases and in controls.However, we have to consider that bone development is not only limited to Ca intake. As mentioned previously, cow’s milk provides several nutrients that are important for body and skeletal health ,31. Of nIn the literature, the majority of data on bone mineralization has been obtained by DXA or quantitative computed tomography (reporting BMD). Recent studies have clarified most of the dubious technical aspects of QUS leading it to its clinical application in a number of disorders ,32,33. NIn our study, no significant association was observed between dietary Ca intake and phalangeal BMS, and the BMS was not different in cases and controls, despite the lower Ca intake. These results are in accordance with previous studies ,42 and iThe inverse correlation between the time spent on a lactose-free diet and the AD-SOS age-related Z-score suggests that a lower Ca intake could affect the BMS on a long period of time. Although the difference in BMS was not significant in the sub-groups (LI versus LT), the AD-SOS age-related Z-score was lower than in the controls.Physical activity seems to be the primary modifiable stimulus for increased bone growth and development in adolescents , accountThe controls spent more time doing extra-curricular exercise compared to the cases, who limited their physical activity to the two hours at school time. Other studies observed a correlation between physical exercise and Ca intake ,47. TheyThe importance of cow’s milk and Ca intake in adolescents is still controversial. This study showed that lactose malabsorbers had a good Ca intake thanks to lactose-free cow’s milk, and a Z-score comparable to the controls.Our results indicate that a lactose-free diet does not affect the phalangeal BMS of adolescents with primary lactase deficiency when their diet includes lactose-free cow’s milk. However, these patients still have a significantly lower intake of Ca compared to peers on a regular diet. The inverse correlation observed between the AD-SOS age-related Z-score and the time spent on a lactose-free diet suggests that a long-term follow up of the malabsorbers is advisable.


High-sensitivity cardiac troponin I (hs-cTnI) has been used in the diagnosis and risk stratification of acute myocardial infarction. However, there is no common consensus on an hs-cTnI reference interval for the Chinese population. The aim of this study was to describe the distribution of hs-TnI and establish the 99th percentile reference interval for hs-cTnI in healthy adults from the Sichuan area.Serum specimens were collected from 1485 healthy adults (731 men and 754 women ranging in age from 18 to 85 years) in Sichuan Provincial People's Hospital. All specimens were divided into 4 groups according to age distribution: 18 to 35 years, 36 to 50 years, 51 to 65 years, and ≥66 years. Specimens were further divided into younger/middle and older-aged groups based on a cut-off age of 50 years. The serum hs-cTnI concentration was determined using the Abbott ARCHITECT STAT hs-cTnI assay.P < 0.05). The 99th percentiles of hsTnI were 28.0 pg/mL among the whole population, 31.1 pg/mL among men, and 22.7 pg/mL among women. The age-dependent 99th percentiles of hs-cTnI in men and women were as follows: 28.8 versus 12.5 pg/mL for 18 to 35 years, 20.4 versus 9.2 pg/mL for 36 to 50 years, 24.2 versus 13.6 pg/mL for 51 to 65 years, and 27.9 versus 32.2 pg/mL for ≥66 years.The serum hs-cTnI concentration increased with age (The 99th percentile reference interval for hs-cTnI in healthy adults from the Sichuan area was similar to the manufacturer's recommendation. Men had a higher 99th percentile hs-cTnI value than women in the age range of 18 to 65 years. Cardiac troponin I is a highly specific indicator of myocyte injury. Therefore, measurement of cardiac troponin I is considered to be highly specific for myocardial injury. International guidelines recommend one concentration measurement of cardiac troponins above the 99th percentile for a healthy reference population with a coefficient of variation of 10% as the cut-off value for the diagnosis of AMI. However, 1st-generation troponins assays were not recommended for the biomarkers of choice in the early diagnosis of AMI due to lack of the recommended detection limit and precision.Cardiovascular disease is the leading cause of morbidity and mortality worldwide. Cardiac troponins are proteins of the contractile apparatus in the heart muscle and are classified as: troponin I, troponin T, and troponin C based on their function. As cardiac troponins are essential components of the contractile apparatus of myocytes, elevation of circulating cardiac troponins concentration reflects injury of the myocardial cells. Cardiac troponins I and T have been identified as the primary biomarkers for the early diagnosis of acute myocardial infarction (AMI) In order to detect the minimal release of troponins, a new generation of high-sensitivity troponins assay has already been introduced into clinical practice. High-sensitivity troponins assays can measure troponins concentrations above the limit of detection in ≥50% of healthy individuals, with a ≤10% coefficient of variation at the 99th percentile. As the sensitivity of troponins assays has improved, these new generation assays can detect the minimal release of cardiac troponins below the 99th percentile, which was often undetectable with the conventional troponins assays. The high-sensitivity cardiac troponin I (hs-cTnI) assay is characterized by a detection limit of concentration in the range of picograms of protein per milliliter. Despite the recommended diagnostic cut-off concentrations for high-sensitivity troponin I (hsTnI), a broad range of cut-off values exist across individual laboratories. A major cause of the broad variation of the 99th percentile cut-off values for hs-cTnI is the selection criteria of the reference population. Males and older persons tend to have higher troponin I values than females and younger persons.–11 In addition, the increasingly analytical sensitivity of new troponins assays requires validation in the 99th percentile estimation.Cardiac troponins assays can be classified as: low, medium, high, and ultrasensitive according to their sensitivity.The normative reference interval for hs-cTnI used in China was determined for healthy Western populations. Considering ethnic diversity, the cut-off value for the 99th percentile hs-cTnI concentration for a Western population might not be applicable for the Chinese population, and ignoring this issue could result in misleading clinical decision-making. Therefore, establishment of a reference interval for hs-cTnI in healthy Chinese adults is necessary. In this study, we aimed to describe the distribution of hsTnI and establish the 99th percentile local reference interval for hs-cTnI in healthy adults from the Sichuan area.22.1 no history of cardiovascular disease; normal liver and renal functions; and without major trauma or cardiac medication history within 3 months. Pregnant women, extreme athletes, those with a chronic disorder requiring regular medication, and those with edema, pleural effusion or ascites, muscle atrophy, cardiac insufficiency, urinary obstruction, or a history of diabetes, hyperlipemia, and hypertension were excluded. Participants were categorized based on age into 4 groups: 18 to 35 years, 36 to 50 years, 51 to 65 years, and ≥66 years. In addition, participants were further divided into younger and older-aged groups according to the cut-off age of 50 years. The study protocol was approved by the Ethics Committee of Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital and followed the ethical principles of the Declaration of Helsinki. All participants provided written informed consent.Between April 17 and April 26, 2015, a total of 1485 health adults seen for check-ups (mean age: 36 ± 13 years) comprising 731 males and 754 females were randomly selected from the Department of Medical Examination Center of Sichuan Provincial People's Hospital. The occupations of the adults spanned government, enterprises, public institutions, schools, and service industries. Randomization was performed by selecting the odd number of the medical record sequence. The inclusion criteria used to define healthy subjects were as follows: normal body mass index 18.5 to 23;2.2g for 10 minutes within 2 hours of collection. Serum samples were stored at −80 °C in a freezer until analysis. Serum levels of total bilirubin, direct bilirubin, indirect bilirubin, alanine aminotransferase, aspartate aminotransferase, total protein, albumin, globulin, alkaline phosphatase, gamma-glutamyltransferase, urea nitrogen, creatinine, uric acid, glucose, triglyceride, total cholesterol, high density lipoprotein cholesterol, and low density lipoprotein cholesterol were measured by the automated ARCHITECT c2000  according to standard clinical laboratory procedures. In addition, demographic characteristics, anthropometric data, heart rate, blood pressure, 12-lead electrocardiogram, chest radiography, and echocardiography were recorded.Three milliliters of blood were obtained from a forearm vein after at least 8 hours of fasting from all participants. After complete coagulation for 120 minutes, blood specimen was centrifuged at 10,000×2/kg were grouped as obese.Blood pressure was measured in the sitting position. Hypertension was defined as a systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg, or the use of antihypertensive agents. Diabetes was diagnosed as fasting blood glucose level ≥7 mmol/L, or the use of insulin or oral any hypoglycemic agents. Participants with a body mass index >30 m2.3Hs-cTnI was measured with the Abbott ARCHITECT STAT hs-cTnI assay (Abbott Diagnostics) on Abott i2000 Automatic Immune Analyzers. Precision for the assay was assessed based on the document of the CLSI EP5-A2. Coefficients of variation at the 99th percentiles were tested with participant's samples that had been diluted with assay diluent.2.4 The nonparametric Mann–Whitney U test was used to compare differences in hs-cTnI values between different groups. A P-value < 0.05 was considered as statistically significant.All analyses were conducted using SPSS 19.0 statistical software  and MS Excel 2010. Categorical data are expressed as number or percentage. Normally distributed data are presented as mean ± SD. The Kolmogorov–Smirnov test was applied to verify the normal distribution of variables. The 99th percentile upper reference limit (URL) based on age and gender was calculated in accordance with the CLSI EP28-A3c guideline using a nonparametric analysis.33.1Figure 3.2P < 0.05). Figure The Kolmogorov–Smirnov test demonstrated the skewed distribution of serum hs-cTnI concentration in different age and gender groups . Men showed a higher serum hs-cTnI concentration than women from age 18 to 65 years (P < 0.05). In the younger and middle-aged groups, the serum hs-cTnI concentration was significantly lower in women than in men (P < 0.05). By contrast, there was no significant gender difference in the serum hs-cTnI concentration in the older-aged group (P > 0.05). The distributions of serum hs-cTnI concentration according to age and gender are shown in Fig. The median values of serum hs-cTnI concentration increased with increasing age across the age groups , Zeller et al (27.0 pg/mL), and Aw et al (25.6 pg/mL). The male 99th percentile cut-off value in our study was slightly higher than the value reported by Krintus et al (27 pg/ml), but our female 99th percentile cut-off value was quite different (22.7 vs 11.0 pg/mL). The healthy populations enrolled in these studies were of sufficient sample size to determine reliable overall and gender-specific 99th percentile values. In another Chinese study conducted in Shanghai, the Abbott 99th percentile value for hs-cTnI was 21 pg/mL for all healthy participants, which was lower than the manufacturer's recommendation of 28 pg/mL. This discrepancy may be related to the different age distribution of the participants. Although the 99th percentile cut-off values for hs-cTnI in our study were close to the recommendations of the Western laboratory, establishing the age-, gender-, and ethnic-specific reference intervals for hs-cTnI is necessary.Cardiac troponins are blood biomarkers that reflect myocardial cell damage. Assays for cardiac troponin I and T have become widely accepted tools in the early diagnosis of AMI. With the improved sensitivity and precision, the latest troponins assays allow the measurement of hs-cTnI in healthy individuals. Different studies have reported different reference intervals even for the same hs-cTnI assay. Differences in study populations and analytical performance of the assay may largely explain the inconsistent findings of the various studies. For studies on the Abbott hs-cTnI assay in healthy individuals, the 99th percentile cut-off value has ranged from 12.3 to 27 pg/mL. Eggers et al investigated the hs-cTnI concentrations in 814 community-dwelling individuals at both 70 and 75 years of age, and they found aging resulted in an increase in the 99th percentile for hs-cTnI value, especially in men. These findings suggest that the optimal cut-off value should be higher for adults over 70 years of age than for younger patients when applying hs-cTnI decision thresholds in clinical settings. An increased hs-cTnI concentration should not be interpreted with isolation. As the hs-cTnI concentration was relatively lower in healthy adults, physiological variation of hs-cTnI may affect the disease diagnosis and treatment. The physiological variation and coefficients of variation of hs-cTnI were 18% and 21%, respectively. The clinical implications of implementation of an hs-cTnI assay should be within the context of a patient's medical history and presentation. Apart from at least one concentration of cardiac troponins above the 99th percentile cut-off, an increase or decrease in cardiac troponins concentration is required for the diagnosis of AMI.In addition to gender-specific differences, we observed a higher median concentration of hs-cTnI among older individuals. Median values of serum hs-cTnI concentration increased with advancing age. Particularly, we observed a sharply increase in the 99th percentile value for hs-cTnI over 66 years in the female subgroup. This finding was supported by a multicenter study in the Italian population. Moreover, the reference population should be selected on the basis of a health questionnaire, screening for renal function and ventricular dysfunction. In this study, we selected 1485 young and elderly adults from the population seeking a routine health check-up who met the criteria of international guidelines.The participant selection criteria had a significant impact on the 99th percentile value. However, defining the appropriate healthy population to determine the 99th percentile is challenging. For a study reporting cardiac troponins with the 99th percentile, at least 300 to 500 individuals should be included. making it unreliable to examine the effect of age on the hs-cTnI concentration. Finally, this study was purely an analytical evaluation of the hs-cTnI assay. Therefore, diagnostic performance of the Abbott hs-cTnI assay needs to be further investigated.Several limitations of this study should be mentioned. First, individuals with subclinical coronary heart disease could not be excluded based on the electrocardiogram and echocardiography. In addition, natriuretic peptides as a surrogate marker for underlying myocardial dysfunction were not investigated in this study. Second, the relatively small sample size in the aged 66 years or older category could not satisfy the recommended minimum number of 300 participants for subgroup analysis,5Using the Abbott hs-cTnI assay, we established 28.0 pg/mL as the 99th percentile value for hs-cTnI in healthy adults from the Sichuan area; this value was close to the manufacturer's recommendation. Men had a higher 99th percentile hs-cTnI value than women at ages 18 to 65 years. The current study supports the notion that individual laboratories need to establish their own age- and gender-specific threshold values for hs-cTnI when defining reference intervals. Establishing a reference interval for hs-cTnI that is suitable for the healthy Chinese population may improve medical decision-making.